WO2021173670A2

WO2021173670A2 - Recombinant antimicrobial peptide as dietary feed supplement for use in improving growth performance and immune response

Info

Publication number: WO2021173670A2
Application number: PCT/US2021/019416
Authority: WO
Inventors: Jyh-Yih Chen
Original assignee: Academia Sinica; Shih, Ming-Che
Priority date: 2020-02-24
Filing date: 2021-02-24
Publication date: 2021-09-02
Also published as: TW202146654A; CN116056587A; WO2021173670A3

Abstract

The present invention relates to a recombinant antimicrobial peptide, which may be expressed in a transformed yeast. The present invention also provides a dietary supplement for improving growth performance and immune response in animals, which comprises the recombinant peptide.

Description

RECOMBINANT ANTIMICROBIAL PEPTIDE AS DIETARY FEED SUPPLEMENT FOR USE IN IMPROVING GROWTH PERFORMANCE AND IMMUNE RESPONSE

FIELD OF THE INVENTION

[0001] The present invention relates to a new recombinant antimicrobial peptide which may be used as a dietary feed supplement for improving growth performance and immune response.

BACKGROUND OF THE INVENTION

[0002] Antibiotics inhibit or kill pathogens that may negatively affect the health of broilers in order to allow maximal growth. Breeders often pursue methods to increase abundance, consistency, and growth speed of animals, including livestock, poultry and aquatic animals. Therefore, control of disease outbreaks by routine administration of antibiotics is a common practice. For many years, antibiotics have been widely used in husbandry industry to reduce bacterial infections [1] However, antibiotic treatments at certain stages of life can severely discompose the microbiota of the intestine, leading to delays in immune system development and disturbances in immune function. For example, inappropriate antibiotic treatment can cause the broilers to become susceptible to pathogen infections at later developmental stages [2] Furthermore, the overuse of antibiotics promotes antibiotic resistance in pathogens, which may then negatively impact animal and human health (https://www.danmap.org/downloads/reports.aspx) [3] The use of antibiotics for promotion of growth has been banned in Europe since 2006 [4, 5]

[0003] Accordingly, new antimicrobial agents that may substitute for antibiotics in poultry production are in demand for use as alternative feed additives. Recently, antimicrobial peptides (AMPs) have emerged as antibiotic equivalents by virtue of their ability to disrupt membrane integrity in bacteria and other pathogens [6] AMPs exhibit powerful antimicrobial activity against different and diverse microorganisms, but typically still have lox hemolytic activity toward host cells [7] Piscidins comprise one of the most widely studied AMP families. Isolated from several fish species, piscidins exhibit an expansive spectrum of biological functions, including antibacterial, antifungal, anti-parasite, anti-nociceptive and antitumor functions [8-12] Five of the piscidin-like AMPs (named TP1 -TP5) from Oreochromis niloticus have been cloned and characterized by some of the study group as the same as the inventors [9] It was revealed that pathogens are less able to develop resistance to picidins than to antibiotics. The toxicity of the piscidin-like AMPs may be due to the relative nonspecificity of electrostatic interactions between piscidins and membrane lipid components of pathogens, such as Klebsiella pneumoniae and Acinetobacter baumannii [13], There are few reports that have described the use of recombinant protein expression systems to produce piscidins [14, 15], [0004] However, there is no way to know whether piscidins are effective in improving growth performance and immune response in chicken. It is still desirable to develop a diet to take the place of antibiotics as feed additives in the domesticated fowl farming industry.

SUMMARY

[0005] Accordingly, the present invention provides a new dietary supplement as a substitute for antibiotics for improving growth performance and immune response in husbandry industry, in which a recombinant Epinephelus lanceolatus piscidin (rEP) is used, to prevent pathogen infection, and to avoid drug-resistance in pathogens.

[0006] According to the invention, the Epinephelus lanceolatus piscidin (EP) comprises an amino acid sequence selected from the group consisting of

(1) EP1 : CIMKHLRNLWN GAK AIYN GAK AGWTEFK (SEQ ID NO: 1),

(2) EP2: CFFRHIKSFWRGAKAIFRGARQGWRE (SEQ ID NO:2), and

(3) EP3: GFIFHIIKGLFHAGRMIHGLVNRRRHRHGMEE (SEQ ID NO:3).

[0007] In one embodiment, the dietary supplement comprises one EP or mixture thereof. In the invention, the Epinephelus lanceolatus piscidin (EP) may be prepared in the form of a feed supplement, which is confirmed to have the effects on growth performance and immune response of G. domesticus (chicken).

[0008] In another aspect, the present invention provides a method for improving growth performance and immune response in an animal, which comprises administering to said animal an effective amount of the rEP of the invention.

[0009] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. [0011] Figure 1 provides the sequences of E. lanceolatus piscidins g6496.tl (EP-1), g6497.tl (EP- 2) and g6498.tl (EP-3); wherein the nucleotide (nt) sequences and predicted amino acid (aa) sequences are shown. Nucleotides are numbered beginning with the first nucleotide. Asterisk (*) indicates a stop codon. The EP cDNA gene (g6498.tl) was modified based on the preferential codon usage of P. pastoris expression system.

[0012] Figure 2 shows the sequence alignment of tilapia piscidins and Epinephelus lanceolatm piscidins. Multiple sequence alignments of the tilapia piscidin peptides with isolated E. lanceolatm piscidins (g6496.tl, g6497.tl and g6498.tl); wherein the gaps were inserted to obtain maximum homology. All coding sequences were input into the dendrogram for alignment. The same amino acid is indicated by the same color, e.g., methionine (M) is indicated by yellow. The result of a phylogenetic analysis of piscidins from tilapia (TP1 to TP5) and E. lanceolatm is shown.

[0013] Figure 3 shows the expression of the E. lanceolatus piscidin-6><His (rEP) protein in Pichia pastoris. Figure 3(a) shows the plasmid map of the pPICZaA-EP-his vector. Figure 3(b) provides the different concentrations of methanol were used for induction, and recombinant protein expression was analyzed by western blotting. Figure 3(c) shows that the cells were harvested and total protein from supernatant and pellet were analyzed by SDS-PAGE and western blotting. Lane 1, low-range rainbow marker; lane 2, synthesized EP; lane 3, protein expressed by the pPICZaA vector; lane 4-9, cells containing EP expression vector after induction for 0 h (no methanol induction), 1, 2, 3, 4 and 5 days.

[0014] Figure 4 provides the effect of medium composition on the expression of rEP in P. pastoris. The effect of nutrient content was evaluated by comparing P. pastoris X33 transformant cultures grown in BMGY (flask culture, circles) and BSM (fermenter culture, squares) according to their (a) wet cell weight and (b) cell counts.

[0015] Figure 5 shows that Antimicrobial activity of rEP produced in P pastoris by flask and fermenter methods. Figure 5(a) shows the rEP concentration in the yeast culture supernatant before and after induction with methanol for 24 h to 120 h in flask culture. After 5 days of induction, yeast culture supernatant produced large inhibition zones, ordered by width, for K. oxytoca , E. coli , P. aeruginosa , and S. aureus. The marker represents Gram-negative; “+” represents Gram-positive. Figure 5(b) shows the antibacterial activity of rEP produced by the fermenter method. Yeast were induced with methanol from 24 h to 120 h in a fermenter. The supernatant showed inhibitory activity toward E. coli, P. aeruginosa, and S. aureus, as measured by OD₆₀₀. The flask supernatant exhibited greater antimicrobial activity compared to fermenter supernatant.

[0016] Figure 6 shows the effects of rEP on Gram-positive and Gram-negative bacteria by coincubation assay. OD₆₀₀ was measured. Lower OD₆₀₀ compared to control indicates that growth was inhibited. Vector control means empty pPICZaA vector. EP means protein expressed by the pPICZaA-EP-his vector.

[0017] Figure 7 shows that the effects of rEP administration in chicken were evaluated by determining the levels of immunological factors, including (a) Tumor necrosis factor (TNF)-a, (b) Interferon (IFN)-γ, (c) Interleukin (IL)- Iβ, (d) IL-6, (e) IL-10, (f) immunoglobin G (IgG), and (g) Lysozyme (Lyz) by ELISA.

[0018] Figure 8 shows the effects of orally administered rEP on posture, body weight, intestinal morphology, and gut flora. Figure 8(a) shows that after 28 days of feeding, the 1.5% rEP -receiving chickens were larger than the spiraline A and basal diet controls. Figure 8(b) provides the representative images of hematoxylin- and eosin-stained intestinal villi and crypts. Figure 8(c) shows that the intestinal villus length and crypt depth were measured. Figure 8(d) shows the effects of feeding 1.5% rEP on body weight. After 28 days of the trial, the body weight of the rEP group increased significantly. Figure 8(e) shows the effects of orally administrated the spiraline A, basal diet, and 1.5% rEP on intestinal microflora in G. g. domesticus. Relative proportions of bacterial families are shown. Total viable counts of Enterobacteria and Staphylococca were reduced, while the abundance of Lactobacillaceae and Enterococcaceae were increased in the duodenum.

DETAILED DESCRIPTION

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as is commonly understood by one of skill in the art to which this invention belongs. [0020] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021] The term “comprise” or “comprising” is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term “comprise” or “comprising” encompasses the term “consists” or “consisting of.”

[0022] As used herein, the terms “around”, “about” or “approximately” can generally mean within 20 percent, particularly within 10 percent, and more particularly within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly indicated.

[0023] As used herein the term “animal” or “animals” refers to a livestock, a poultry or an aquatic animal.

[0024] The present invention provides a new dietary supplement, which may be used as a feed, and mixed with antibiotics. In the invention, the survival and prevent disease outbreaks were improved after the administration to animals, including livestock, poultry and aquatic animals, to avoid the overuse of antibiotics, which may promote the development of antibiotic-resistant bacteria.

[0025] In the present invention, a new recombinant Epinephelns lanceolatus piscidin (EP) is provided, which is confirmed to have the effect on the growth performance in Gallus gallus domesticus (chicken).

[0026] In the present invention, the gene encoding EP was isolated, sequenced, codon-optimized and cloned into an expression system. The expressed recombinant EP was tested to be used as a dietary supplement for G. g. domesticu ; overall health, growth performance and immunity. The supernatant of the rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 μg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation significantly increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-g production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus , serving to enhance growth and health of the animals.

[0027] Accordingly, the present invention provides a dietary supplement which is the extract from the culture for expressing AMPs. In one embodiment of the invention, the dietarty supplement may be prepared in the form of a fodder supplement in agriculture.

[0028] In the present invention, three cDNAs encoding putative antimicrobial piscidin peptides were isolated from E. lanceolatus and characterized. The transcripts were named g6496.tl (encoding for EP-1), g6497.tl (encoding for EP-2) and g6498.tl (encoding for EP-3), and respectively encode putative AMPs of 76, 76, and 69 amino acid residues. Based on sequence alignments between tilapia piscidins and the EP1 (g6496.tl), EP2 (g6497.tl) and EP-3 (g6498.tl), it was found that EP-3 (g6498.tl) exhibits high similarity to the highly active TP3 and TP4 peptides from tilapia.

[0029] In the examples of the invention, it is suggested that the peptide EP-3 (g6498.tl) had better activity than EP-1 (g6496.tl) or EP-2 (g6497.tl), which also possess antimicrobial or growth inhibition activity in Gram-negative and Gram-positive bacteria.

[0030] In the present invention, the EP was expressed using a gene with optimized yeast codons in the pPICZaA expression vector for P. pastoris [21]. Since EP is a fish gene, it is expected that optimization of the gene for Pichia codon usage would increase yield. Notably, other researchers have reported difficulties in producing high levels of AMPs due to degradation by host proteolytic enzymes [15], In the present invention, it was not found that proteolysis prevented substantial accumulation of rEP in Pichia. His-tagged rEP expressed in P. pastoris showed antimicrobial activity against Grampositive and Gram-negative bacteria.

[0031] In the present invention, the EPs from E. lanceolatus that were expressed in P. pastoris , were found to be relatively stable in vitro after incubation in extreme temperature conditions (100°C), still retaining some antimicrobial activity against S. aureus (BCRC 10780). The stability of rEP may be related to its high arginine content. In line with this notion, it was previously reported that increasing arginine composition of peptides increases antimicrobial activity and enhances the ability of peptides to embed into membranes [22]. Increased arginine content may also enhance the translocation and membrane permeabilization functions [23].

[0032] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0033] Examples

[0034] 1. Materials and Methods

[0035] 1.1 Animals

[0036] All procedures involving animals were conducted in accordance with the requirements of National Pingtung University of Science and Technology (NPUST), and were approved by the Animal Care and Use Committee of NPUST (NPUST- 107-026).

[0037] 1.2. Molecular cloning, construction and transformation of expression plasmid, and selection of positive transformants

[0038] The sequences of primers used to amplify EP were designed according to a previous transcriptome analysis and experimental method [9, 16], Briefly, piscidins were isolated from E. lanceolatus by RT-PCR. mRNA was extracted from E. lanceolatns liver and reverse transcribed; primers used to amplify cDNA are listed in Table 1. Three recombinant clones (g6496.tl, g6497.tl, g6498.tl) were chosen for sequencing.

Table 1 List of primer sequences

¹ g6496_t l : Epinephelus lanceolatus piscidin-1 2g6497.tl : Epinephelus lanceolatus piscidin-2 3g6498.tl : Epinephelus lanceolatus piscidin-3

[0039] Multiple sequence alignments of the clones were made with E. lanceolatus and 0. niloticus piscidin peptides. The following peptides were synthesized by GL Biochem Ltd. (Shanghai, China), and antimicrobial activity of each peptide was determined as minimum inhibitory concentration (MIC) from a microbroth dilution series [9, 13]:

(1) EP-l: g6496.tl (Ac-CIMKHLRNLWNGAKAIYNGAKAGWTEFK-NH2),

(2) EP-2: g6497.tl (Ac-CFFRHIKSFWRGAKAIFRGARQGWRE-NH2), and

(3) EP-3: g6498.tl (Ac-GFIFHIIKGLFHAGRMIHGLVNRRRHRHGMEE-NH2).

[0040] A 102-bp codon-optimized sequence corresponding to the mature EP cDNAgene (g6498.tl) was designed. The sequence was based on the preferential codon usage of Pichia pastoris according to the Graphical Codon Usage Analyser (http://gcua.schoedl.de/) and synthesized by Omics Bio (Taipei, Taiwan). The DNA was cloned into EcoRI/Xbal-digested pPICZaA by Omics Bio (Taipei, Taiwan) to create the recombinant vector, pPICZaA-EP-his. The EP cDNA gene (g6498.tl) sequence in the vector was then confirmed by sequencing.

[0041] The pPICZaA-EP-his was linearized by SacI and then transformed into P. pastoris X-33 by electroporation (1.5 kV, 25 pF, 200 W; ECM 399 electroporation system, BTX Harvard Apparatus). Identification of positive transformants and selection of a rEP-expressing clone with high expression was performed as previously reported, without modification [17] Briefly, the recombinant P. pastoris strain containing the gene for rEP was incubated in 3 ml YPD medium (with 300 μg Zeocin). After 24 h (30°C), 1 ml P. pastoris was used to inoculate 30 mL BMGY medium for 24 h (30°C).

[0042] The Cells were then harvested by centrifugation at 6000 *g for 15 min. The cell pellet was resuspended in 50 mL BMMY medium. The supernatant and pellet were examined by SDS-PAGE and Western blot using His-tag antibody (Abeam, ab213204). The recombinant protein was identified by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry. The antimicrobial activity of rEP was analyzed using an inhibition zone assay at different treatment temperatures [18] All assays were performed in triplicate.

[0043] 1.3. Expression of rEP in a fermenter

[0044] To optimize the expression conditions in a 5000 ml fermenter, a single rEP colony was used to inoculate 200 ml BMGY with PTM4 medium for 36 h at 28°C, 200 rpm. The culture was then transferred to a 5000 ml fermenter (Winpact, Major Science, Taoyuan, Taiwan) containing 3000 ml commercial fermentation medium (BMGY with PTM4) [19] During fermentation, the temperature was maintained at 30°C. The pH was adjusted to 6.0 with 14% ammonia and 0.1 N H₂SO₄, and dissolved oxygen was maintained above 20% saturation. To produce rEP for supplementation of G. g. domesticus fodder, we expressed rEP in a fermenter using basal salt medium and PTM1 [20] After glycerol was completely consumed (19 h), 50% w/v glycerol was fed for 360 min. Next, a 100% methanol feed was started and maintained for 24-96 h. The rEP-expressing yeast cultures were then centrifuged at 6000 rpm for 30 min, and supernatants were spray-dried (YC-500, Shanghai Pilotech Instrument & Equipment Co, Ltd.) before mixing into fodder. Before fodder production, rEP expression in each culture was validated by Western blotting, and the rEP content was assessed by comparing with synthetic EP peptide.

[0045] 1.4. Antimicrobial activity of rEP and fodder preparation

[0046] The antimicrobial activity of rEP was tested on cultures of Staphylococcus aureus (BCRC 10780), Escherichia coli (BCRC 10675), Pseudomonas aeruginosa (ATCC 19660), and Riemerella anatipestifer (RA3, RA9, RA16, CFC27, CFC363, CFC437). The bacteria cultures were derived from single clones that had been expanded and stored at -70°C. The cultures were inoculated in liquid media and cultured overnight at 37°C on a shaker at 180 rpm. Subsequently, the bacteria were diluted in fresh medium (1:1000) and incubated under the same conditions. The four types of bacteria (10⁴ CFU/ml) were mixed with 100 μl of the rEP solution and incubated overnight at 37°C. Supernatant from P. pastoris transformed with vector alone was used as a control. After 24 h, culture growth was assessed by OD₆₀₀. All assays were performed in triplicate. The composition analysis of diets fed to G. g. domesticus is shown in Table 2.

Table 2 Proximate analysis of basal feed with additive and commercial feed composition

[0047] 1.5. G. g. domesticus maintenance and dietary treatment

[0048] A total of 189 male or female 2-day-old G. g. domesticus were randomly assigned to seven dietary treatment groups, including rEP (0.75, 1.5, 3.0, 6.0 and 12%), spiraline-A (Juily Pharmaceutical Co., LTD. Sanxia Dist., New Taipei City, Taiwan), and basal diet groups. Each treatment group consisted of one cage with twenty-seven G. g. domesticus. The composition of the fodder is presented in Table 3-5.

Table 3 Formulation of the basal diet¹ of early stage

Table 4 Formulation of the basal diet¹ of middle stage

Table 5 Formulation of the basal diet¹ of late stage

¹ Fermentation supernatant spray dry powder was added to diets at the expense of cellulose to provide concentration of 0. 0.75 1.5, 3.0, 0.0 and 12 g/100 g diet.

² Antibiotic: each, gm contains: Spiramycin Adipate 30mg(pot.) Streptomycin Sulfate 30mg(pot.) Vitamin A 2.500I.U. Vitamin B1 5mg Vitamin B2 10mg Vitamin B 62mg Vitamin B12 dmcg Vitamin E 2mg Vitamin D3 500I.U. Vitamin K4 1mg Folic Acid 0.2mg Calcium Pantothenic Acid 5mg Nicotnic Acid 10mg Lysine 20mg

[0049] The animals were allowed ad libitum access to fodder and water for the duration of the trial, beginning from 3 days of age. The total time of feeding was 35 days. The G. g. domesticus were maintained in a cage (animal container) with a constant environment (28.9-37.8°C, 12/12 h light-dark cycle). rEP fodder and fecal sewage were not directly discharged to the outside.

[0050] 1.6. Sample collection and Enzyme-linked immunosorbent assay

[0051] Body weight and survival rate were monitored daily during the experimental period. The weight gain, feed efficiency (FE), protein efficiency ratio (PER), and survival percentage were calculated. After 35 days of treatment, the G. g. domesticus were euthanized, blood samples were collected and serum was obtained by centrifugation (3000 xg for 15 min at 4°C). Serum was kept at -80°C until analysis. Enzyme-linked immunosorbent assay (ELISA) was performed in accordance with the manufacturer's standard procedures to determine the levels of immunological factors, including TNF-a, Interleukin-1 b, Interleukin-6, Interleukin- 10, Lysozyme, Immunoglobin-G (IgG), and Interferon-g. Chicken serum samples were analyzed with ELISA kits from ABclonal Inc. (Woburn, MA, USA). After the reaction was completed, the optical density (OD) was measured at 450 nm on a microplate reader (SpectraMax® i3, Molecular Devices, Lagerhausstrasse, Wals, Austria).

[0052] 1.7. Statistical analysis

[0053] Data were analyzed with Prism 7 software (GraphPad Inc., La Jolla, CA, USA). Values represent the mean ± standard deviation (SD). P < 0.05 was considered significant for one-way

Analysis of variance (ANOVA) with Tukey's multiple comparison test.

[0054] 2. Results

[0055] 2.1. Novel piscidins from E. lanceolatus exhibit antimicrobial activity

[0056] The cDNA coding regions for different piscidin sequences (SEQ ID NO: 10, 12 and 14; Figure 1) were isolated and characterized from E. lanceolatus. The three identified cDNA sequences were named g6496.tl, g6497.tl and g6498.tl, and respectively encoded 76, 76 and 69 amino acids (SEQ ID NO: 11, 13 and 15). Alignment of E. lanceolatus piscidins (g6496.tl, g6497.tl, and g6498.tl) and Oreochromis niloticus piscidins (TP1-TP5) revealed remarkably, high sequence similarities (Figure 2). A phylogenetic tree showed that g6498.tl corresponded to TP4 and TP3, which exhibit the best antimicrobial activity characterized to date (Figure 2).

[0057] Next, antimicrobial activity was determined for synthetic g6496.tl, g6497.tl and g6498.tl peptides. All three peptides were active against Gram-positive and Gram-negative bacteria, as shown in Table 6.

Table 6 In vitro activity of antibacterial peptides against Gram-positive and Gram-negative bacteria.

¹g6496.tl: Epinephelus lanceolatus piscidin-1 (EP-1) ²g6497.tl : Epinephelus lanceolatus piscidin-2 (EP-2) ³g6498.tl : Epinephelus lanceolatus piscidin-3 (EP-3)

(the MIC units are in μg/ml)

[0058] The g6498.tl and g6497.tl peptides were also active against Methicillin-resistant Staphylococcus aureus (MRS A), with 5.6 μg/ml and 60 μg/ml MICs, respectively. The two peptides were also toxic to Vibrio parahaemolyticus at similar MICs. Since g6498.tl had stronger antimicrobial activity than g6496.tl and g6497.tl, we named this peptide EP and further produced it in a P. pastoris protein expression system.

[0059] 2.2. P. pastoris expression system for rEP peptide

[0060] As shown in Figure 3(a), the constitutive expression vector pPICZaA-EP-his contains a methanol-inducible AOX promoter, an a-factor signaling peptide, and the STE13 gene for Dipeptidyl aminopeptidase A. The amino acid sequence for rEP was encoded by a gene insert that is codon- optimized for E pastoris. P. pastoris (X-33) transformants were grown on a Zeocin plate (25 μg/ml) and screened by colony hybridization with anti-His antibody; clones with high expression were selected for subsequent experiments. After identification of clones, we validated the presence of a correct expression cassette by PCR amplification and DNA sequencing. During the initial expression experiments, the optimal methanol concentration was determined by supplying different concentrations of methanol for 24 h. The supernatants and pellet (after centrifugation) were collected and analyzed by Western blotting. The results showed that 1% methanol induced robust expression of rEP (Figure 3(b)). The time-dependent effects of 1% methanol induction are shown in Figure 3(c). Thus, 1% methanol induction was selected for subsequent fermenter experiments.

[0061] 2.3. Effects of time and medium on fermenter production of rEP

[0062] To evaluate the effect of induction time and medium on rEP expression, transformants were grown in a fermenter with basal salt medium (BSM) or BMGY. Large-scale production of rEP from P. pastoris (X-33) may be most efficient with high-density cultivation methods. Thus, rEP induction was evaluated in a 5000 mL fermenter. When the wet weight of cells reached 200 g/L (16-18 h postinoculation) in a glycerol-fed batch, 1% methanol was added for times ranging from 24 to 120 h. After methanol induction, the yeast number decreased (Figure 4(a) and 4(b)). The total rEP protein levels reached maxima of 0.8 mg/L in supernatant and 5.63 mg/L in pellet after 48 h induction in BSM medium. On the other hand, the total maximum rEP protein levels in BMGY medium were 3.6 mg/L in supernatant and 7.74 mg/L in pellet at 48 h. The antimicrobial activity of rEP was also monitored, gradually increasing to its highest level after 120 h methanol induction (Figure 5(a), Figure 5(b), Figure 6).

[0063] 2.4. Antimicrobial activity of EP

[0064] The antimicrobial activity of rEP from flask cultures was evaluated by determining its ability to prevent growth of Gram-positive and Gram-negative bacteria with a disk diffusion assay. Tables 7 and Table 8 show the antimicrobial activity and effect of temperature of rEP. The rEP was produced in P. pastoris using a shake flask. The supernatant was collected 120 h after induction and applied to disk paper at the maximum carrying capacity, wherein the disk paper was placed on a bacterial culture plate for 16 h at 37°C. Inhibition zone diameters were measured. Representative radial diffusion assays. rEP exhibited broad-spectrum antimicrobial activity against the tested bacterial strains. The antimicrobial activity and effect of temperature of rEP. The rEP was produced in P. pastoris using a shake flask. The supernatant was collected 120 h after induction and applied to disk paper at the maximum carrying capacity, wherein the disk paper was placed on a bacterial culture plate for 16 h at 37°C. The inhibition zone diameters were measured. Representative radial diffusion assays. The most susceptible pathogens were R. anatipestifer (CFC27) and R. anatipestifer (CFC437) (Table 7). Next, we investigated the thermostability of flask-fermented rEP against S. aureus (BCRC 10780), E. coli (BCRC 10675) and P. aeruginosa (ATCC 19660). Empty vector with no insert in P. pastoris was used as a control. Thermostability was measured by disk diffusion assay after cultures were incubated for 5 min at 40, 60, 80, or 100°C (Table 8).

Table 7 The antimicrobial activity of rEP

Table 8 The effect of temperature on rEP antimicrobial activity

¹NI, no inhibition. ² Vector control was fermentation supernatant from transformants harboring empty plasmid. Ampicillm (2 mg/mi) was applies at a volume of 10 μ1 to disk paper.

[0065] We found that the inhibition zone diameter (mm) values were decreased by increasing pretreatment temperature, but the antimicrobial activity was not affected by different temperatures in Staphylococcus aureus (BCRC 10780). This finding suggests that the tertiary (or secondary) structure of rEP is important for peptide stability, and it should be protected from high temperatures to maintain activity

[0066] 2.5. rEP supplementation improves growth performance and immune response

[0067] The growth performance of G. g. domesticus was evaluated in terms of weight gain and feed efficiency (FE). At the end of the 35-day experimental period, animals fed with 1.5% and 3.0% rEP had significantly higher growth performance than those in the spiraline-A and basal diet groups (Table 9). The physiological effects of daily rEP administration in chicken were then evaluated by determining the levels of immunological factors in the serum, including immunoglobin G (IgG), Tumor necrosis factor (TNF)-a, Interleukin (IL)- 1 β, IL-6, IL-10, Lysozyme (Lyz) and Interferon (IFN)-γ by ELISA (Figure 7(a)-7(g)). No significant changes between groups were observed for TNF- α, I1-1β, 11-6 or Lyz levels (Fig. 7a, c, d, and g). The group supplemented with 1.5% EP showed significantly increased IFN-g level compared to the antibiotics and control groups (Figure 7(b), P = 0.0043 compared to antibiotic group, and P = 0.0073 compared to the control group). In addition, the IL-10 level was significantly higher in the rEP-supplemented group than in the control group ( P = 00.0341) but was not different than the antibiotic group (Figure 7(e)). Chicken fed with antibiotics showed significantly higher serum IgG levels compared to the control group (P = 0.0394) (Figure

7(f)).

Table 9. Weight gain, feed efficiency (FE), protein efficiency ratio (PER) and survival of Gallus gallus domesticus fed with diets containing rEP (0.75, 1.5, 3.0, 6.0 and 12%) of fermentation supernatant spray-dried powder for 4 weeks¹

¹ Values in the same column with different superscript are significantly different (p<0.05). Data are expressed as mean ± SD from group of chicken (n = 27).

² Weight gain (%) = {Filial body weight (g) - Initial body weight (g) }/ Initial body weight (g) x 100 3 Feed efficiency = {Final body weight (g) - Initial body weight (g)}/ Feed intake (g) ⁴ Protein efficiency ratios = {Final body weight (g) - Initial body weight (g)}/ Protein intake (g)

[0068] Given the above, it was found in the present invention that the rEP improved the growth performance of G. g. domesticus compared to Spiraline-A-supplemented fodder. These results suggest that the EP can potentially replace antibiotics to improve growth performance (Figure 8(a) and 8(d)). Histological analysis showed that duodenum villus height was significantly greater in rEP animals than that in the control group (Figure 8(b) and 8(c)). In the present invention, it was found that 1.5% rEP significantly increased the numbers of Lactobacillaceae and Entercoccaceae, while decreasing the numbers of Enterobacteriaceae and Staphylococcaceae in duodenum when compared to spiraline A and basal diet groups (Figure 8(e)). It was found that no Salmonella could be detected in blood after challenge (data not shown). After daily rEP administration, IL-10 was significantly higher in the rEP- supplemented group compared to the control group. IL-10 is a proinflammatory cytokine, which is produced by different cells, such as Th2 cells, macrophages and monocytes, and functions as an immunoregulator during infection with bacteria, fungi and viruses. The higher IFN-g production in rEP-supplemented animals may increase cytokines that induce antimicrobial pathways to protect against extracellular and intracellular pathogens.

[0069] It is concluded that the recombinant EP possesses the beneficial use as a supplement that may replace antibiotics in G. g. domesticus feed. The results indicate that supplementation of 1.5% or 3% rEP in chicken fodder can improve growth performance, intestinal morphology, microbiota and immunity in broilers, and other animals.

[0070] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

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Claims

What is claimed is:

1. A recombinant Epinephelus lanceolatus piscidin (EP), which comprises an amino acid molecule consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.

2. The EP according to claim 1, which is an amino acid molecule consisting of the amino acid sequence consisting of SEQ ID NO:l.

3. The EP according to claim 1, which is an amino acid molecule consisting of the amino acid sequence consisting of SEQ ID NO:2.

4. The EP according to claim 1, which is an amino acid molecule consisting of the amino acid sequence consisting of SEQ ID NO:3.

5. A dietary supplement for improving growth performance and immune response in chicken, which comprises the EP as defined in any one of claims 1-4.

6. The dietary supplement of claim 5, which is in a form of a feed supplement.

7. A feed supplement for improving growth performance and immune response in chicken, which comprises the EP as defined in any one of claims 1-4.

8. A method for improving growth performance and immune response in an animal, which comprises administering to said animal an effective amount of the EP as defined in any one of claims 1-4.

9. The method of claim 8, wherein the animal is a livestock, a poultry or an aquatic animal.

10. A use of a composition in improving growth performance and immune response in an animal, in which the composition comprises an effective amount of the EP as defined in any one of claims 1-4.

11. The use of claim 10, wherein the animal is a livestock, a poultry or an aquatic animal.